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Distant non-obvious mutations influence the activity of a hyperthermophilic pyrococcus furiosus phosphoglucose isomerase

DOI: 10.3390/biom9060212 DOI Help

Authors: Kalyanasundaram Subramanian (Wageningen University) , Karolina Mitusińska (Silesian University of Technology) , John Raedts (Wageningen University) , Feras Almourfi (King Abdulaziz City for Science and Technology (KACST)) , Henk-jan Joosten (Bio-Prodict) , Sjon Hendricks (Wageningen University) , Svetlana E. Sedelnikova (University of Sheffield) , Servé W. M. Kengen (Wageningen University) , Wilfred R. Hagen (Delft University of Technology) , Artur Góra (Silesian University of Technology) , Vitor A. P. Martins Dos Santos (Wageningen University) , Patrick Baker (University of Sheffield) , John Van Der Oost (Wageningen University) , Peter J. Schaap (Wageningen University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biomolecules , VOL 9 , PAGES 212

State: Published (Approved)
Published: May 2019
Diamond Proposal Number(s): 1218

Open Access Open Access

Abstract: The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

Journal Keywords: Protein engineering; Comulator; cupin phosphoglucose isomerase; Pyrococcus furiosus; solvent access

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Documents:
s41467-019-10714-y.pdf